Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for the lipogenic marker genes PLIN2 and PPARg , as well as COL1A1 in human lung fibroblasts treated with metformin or vehicle. (E, F) Staining of lipid droplets in fibroblasts using LipidTOX (red). Nuclei were counterstained with DAPI (blue). (G-H) Gating strategy for detecting LipidTOX + cells by flow cytometry. (I) Quantification of LipidTOX + cells in response to metformin treatment. (J) Heatmap representation of the top 100 differentially expressed genes in fibroblasts following metformin treatment. Scale bars: (E, F) 25 µm. (B-D) Each data point within a given group corresponds to one patient. Vehicle-treated group: n=12, Metformin-treated group: n=11. (I) n=3 per group. ** P<0.01, *** P<0.001, **** P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Marker, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72h. (E-H) Staining of TGFβ1- and vehicle-treated cells with LipidTOX (red), anti-ACTA2 antibodies (green) and DAPI (blue). (I-K) qPCR analysis for PLIN2, PPARg and COL1A1 in human lung fibroblasts treated with TGFβ1 or vehicle for 72 h, followed by treatment with metformin or vehicle for 72 h. (L-M) Staining of TGFβ1-and vehicle-treated cells with LipidTOX (red) and DAPI (blue) at the end of treatment (t=144 h). Scale bars: (E-H) and (L-M) 25 µm. (B-D, I-K) Each data point within a given group corresponds to one patient. (B-D) n=4 per group. (I-K) n=9-10 per group. * P<0.05, **P<0.01, ****P<0.0001.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-E) Bright-field imaging of PCLS treated with metformin or vehicle for five days. (F, G) Hematoxylin and eosin staining and COL1A1 immunostaining of PCLS prepared from a non-IPF donor lung. (H-M) Hematoxylin and eosin staining, Masson’s trichrome staining and COL1A1 immunostaining of PCLS prepared from an IPF lung and treated with metformin or vehicle for five days. (N, O) 3D-reconstruction of z-stacks of metformin- and vehicle-treated PCLS stained for COL1A1 (green) and lipid droplets (red). (P) Gating strategy for flow cytomety-based quantification of LipidTOX + cells that are negative for hematopoeitic (CD45), endothelial (CD31) and epithelial (EpCAM) cell markers. (Q) Quantification of flow cytometry measurements on metformin- and vehicle-treated cells. (R) Total collagen assay for metformin- and vehicle-treated cells. Scale bars: (B-E) 2 mm, (F) 500 µm, (G, L, M) 50 µm, (H-K) 200 µm. (Q, R) Each data point within a given group corresponds to one patient. (Q) n=4 per group. ® n=3 per group. * P<0.05, **P<0.01.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Imaging, Staining, Immunostaining, Flow Cytometry, Collagen Assay

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the Acta2-Cre-ERT2 and tdTomato flox construct. (B) Schematic representation of the timeline of the experiment. Bleomycin was administered intratracheally at day 0. Between days 5 and 14, mice were fed tamoxifen-containing pellets and starting at day 14, metformin (1.5 mg/mL) or vehicle was administered through drinking water. Mice were sacrificed at day 28. (C-F) Hematoxylin and eosin and Masson’s trichrome staining of metformin- and vehicle-treated lungs. (G) Quantification of fibrosis in metformin- and vehicle-treated lungs. (H, I) Immunofluorescence for COL1A1 (green). Endogenous tdTomato signal (red) and DAPI (blue) are also shown. (J) LipidTOX staining (green) and tdTomato + cells (red) are shown. The box in (J) is magnified in (K). Arrowheads indicate LipidTOX + tdTomato + cells. (L-S) Gating strategy (to detect CD45 - CD31 - EpCAM - tdTomato + and/or LipidTOX + cells) and quantification of various cell populations based on tdTomato and LipidTOX detection. Scale bars: (C-F) 1 mm, (H, I) 50 µm, (J) 25 µm. (G, Q-S) Each data point within a given group corresponds to one animal. n=5 per group. * P<0.05, **P<0.01. IF: Immunofluorescence, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Construct, Staining, Immunofluorescence

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the gain-of-function experimental setup for AMPK signaling. (B-E) qPCR analysis of PLIN2, PPARg , COL1A1 and BMP2 in IPF fibroblasts treated with AMPK agonist GSK621 or vehicle. (F) Schematic representation of the loss-of-function experimental setup for AMPK signaling. (G-I) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with AMPK siRNA or scramble siRNA. The decrease of AMPK protein levels at the time of analysis is shown in (J, K). (L-N) Staining of GSK621- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). Metformin-treated cells were used as a positive control for lipid-droplet accumulation (M). Scale bars: (L-N) 25 µm. (B-E, G-I, K) Each data point corresponds to one patient. (B-E) Vehicle-treated group: n=7-8, GSK621-treated group: n=6-8. (G-I) n=4 per group. (K) n=3 per group. * P<0.05. ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Positive Control

Journal: bioRxiv

Article Title: Metformin induces lipogenic differentiation in myofibroblasts to reverse mouse and human lung fibrosis

doi: 10.1101/401265

Figure Lengend Snippet: (A) Schematic representation of the experimental setup. (B-D) qPCR analysis of PLIN2, PPARg and COL1A1 in IPF fibroblasts treated with rhBMP2 or vehicle. (E, F) Staining of rhBMP2- and vehicle-treated cells with LipidTOX (red) and DAPI (blue). (G) Western blot showing the induction of PPARγ phosphorylation in response to rhBMP2 treatment. Lanes 1-4 and lanes 5-8 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (H) Western blot showing the opposing effects of metformin and TGFβ1 on PPARγ phosphorylation, and the ability of metformin to partially restore PPARγ phosphorylation in TGFβ1-treated cells. Lanes 1-12 and lanes 13-18 were run in parallel on different gels under the same conditions. Quantification of the immunoblot is shown in the lower panel. (I) Model for the antifibrotic mechanism of action of metformin in human lung fibrosis. Metformin activates AMPK signaling in myofibroblasts, leading to suppression of collagen production, and induces lipogenic differentiation via an AMPK-independent mechanism involving BMP2 release and PPARγ activation. Arising lipofibroblasts are known to support type 2 alveolar epithelial stem cells in the lung. Scale bars: (E-F) 50 µm. (B-D, G, H) Each data point corresponds to one patient. (B-D) n=10-11 per group. (G) n=4 per group. (H) n=3 per group. * P<0.05, ns: Not significant.

Article Snippet: Anti-collagen 1 A1 (anti-COL1A1) antibodies (Rockland, 1:200) and goat anti-rabbit antibodies (Life Technologies, 1:500) were used for immunofluorescence.

Techniques: Staining, Western Blot, Activation Assay